This invention is in the field of pharmacology, and relates to human use of a yellow pigment called zeaxanthin (ZX) in preventing or treating macular degeneration, a disease which damages retinal tissue and causes blindness.
This application is being simultaneously filed with a related patent application entitled, xe2x80x9cMethod of Making Pure 3R-3xe2x80x2R Stereoisomer of Zeaxanthin for Human Ingestion,xe2x80x9d Ser. No. 08/551,166. Both of these two patent applications are assigned to the same assignee (Applied Food Biotechnology, Inc. of O""Fallon, Mo.). The contents of that related application are incorporated herein by reference; upon issuance of a US patent based upon this current application, that related application will be opened for public inspection, if it has not already issued as a US patent.
In addition to describing methods of manufacturing and purifying commercially useful quantities of isomerically pure R-R zeaxanthin, that related patent application contains a fairly extensive discussion of retinal physiology and carotenoid chemistry. Although that Background information will not be repeated herein it its entirety, an overview is provided in the next four paragraphs, to help introduce and explain this invention to those who have not read the other related patent application.
Briefly, there is a yellow region called the macula in the central area of the retina, inside the eyeball. The yellow color is caused by two carotenoid pigments, lutein and zeaxanthin. These carotenoids have a yellow color because they absorb the high-energy radiation of the near-ultraviolet and blue light spectrum and reflect the yellow/yellow-orange wavelengths. It is theorized that since these two pigments absorb wavelengths in the high-energy spectrum, they may help protect retinal cells in the macula against xe2x80x9cphototoxicxe2x80x9d damage caused by short-wavelength high-energy light radiation.
Lutein and zeaxanthin are chemically very closely related to each other; both have the exact same chemical formulae, differing only in their ring stereochemistry and the spatial placement of one end ring and the placement of a double bond in that end ring, as shown in FIG. 1.
xe2x80x9cMacular degenerationxe2x80x9d is a medical term that applies to any of several disease syndromes which involve a gradual loss or impairment of eyesight due to cell and tissue degeneration of the yellow macular region in the center of the retina. Age-related macular degeneration (AMD) is the most common form of this type of disease. AMD affects millions of Americans over the age of 60, and is the leading cause of new blindness among the elderly. It is characterized and usually diagnosed by the presence of elevated levels of two types of cellular debris within the retina, called drusen and lipofuscin. These types of cellular debris may accumulate to abnormal levels for a number of reasons, including: (1) retinal cell damage caused by repeated exposure to too much light; (2) inherited genetic factors; (3) poor overall health of an individual; and (4) insufficient quantities of anti-oxidant compounds such as vitamins A, C, and E and selenium in a person""s diet. Accumulation of drusen occurs within the capillaries and in the Bruck""s membrane, and can impede the transport of oxygen and nutrients to the retinal tissues, and the removal of metabolic wastes from the tissues. Accumulations of lipofuscin occurs within a cellular layer which underlies the photoreceptors and which is responsible for nourishing, replenishing and removing wastes from these highly active visual cells. Accumulation of one or both of these types of debris can disrupt the normal metabolic and cellular processes which must occur in order to maintain retinal and visual health.
Although the presence and the apparent or likely protective role of zeaxanthin in the retina have been recognized for more than a decade, no one has previously been able to create purified zeaxanthin preparations suitable for human consumption, either as drugs for treating macular degeneration, or as vitamin/nutritional supplements for reducing the risk of macular degeneration later in life. This has been a major shortcoming, since there are no other effective means for treating or preventing macular degeneration; although xcex2-carotene, vitamin A, and vitamin E have generally beneficial anti-oxidant effects and may slightly retard the rate of macular degeneration, they do not rise to the level of truly effective treatments. For practical purposes, macular degeneration must be regarded as unpreventable, unstoppable, and irreversible, and anti-oxidants such as xcex2-carotene, vitamin A, and vitamin E are merely palliative measures that can be used to try to slow down the encroaching damage somewhat, since nothing better is available to the public.
The primary (and previously insurmountable) problems that have been encountered in prior efforts to create purified forms of zeaxanthin for human ingestion include: (1) the extremely high level of chemical similarity between zeaxanthin and other less desirable carotenoids, including lutein and xcex2-carotene, make it extremely difficult to separate zeaxanthin from lutein and xcex2-carotene on any commercial scale; and, (2)zeaxanthin itself has three different stereoisomers, called the 3R-3xe2x80x2R isomer (which is desirable, and which is present in retinal cells), the 3S-3xe2x80x2S isomer (which is undesirable and which is not believed to exist naturally in retinal cells) and the S-R meso isomer. The S-R meso isomer is not normally ingested in the human diet, and is not present in human blood, but it is sometimes found in the retina, apparently as a conversion product that is sometimes created when lutein is chemically altered by high-energy light waves. Although hard scientific evidence of its roles and effects in the retina is not yet available, the S-R meso isomer of zeaxanthin is presumably less desirable than the naturally occurring R-R isomer.
It is effectively impossible to separate these three isomers of zeaxanthin from each other in commercial quantities; therefore, synthetic methods of creating racemic (mixed-isomer) mixtures of zeaxanthin are effectively useless in efforts to create zeaxanthin for human ingestion. In addition, although it is theoretically possible to chemically synthesize enriched formulations having reduced quantities of the undesired S-S and S-R isomers, the necessary chemical techniques are extremely difficult and expensive, and have never been commercialized.
That completes an overview of the scientific and medical background of this invention: as mentioned above, these topics are discussed in more detail in the related patent application cited above.
In analyzing the prior art, it must be recognized that zeaxanthin has been known for more than a decade to be present in the retina, and scientists have hypothesized for at least that long that zeaxanthin appears to play a beneficial protective role in helping prevent phototoxic damage in the retina. However, no one prior to this invention has ever been able to synthesize the purified R-R stereoisomer of zeaxanthin in any quantities sufficient for human consumption. This failure can be attributed to the extraordinary difficulties of (1) separating zeaxanthin from other carotenoids, and (2) isolating the desired R-R isomer of zeaxanthin and removing the undesirable S-S and S-R isomers.
Accordingly, As of October 1995, the only way to purchase zeaxanthin, either in purified form or in a semi-concentrated form in which zeaxanthin comprises more than about 5% of the weight of the preparation, requires the purchase of very small quantities of zeaxanthin (measured in milligrams) from specialty chemical manufacturers such as Atomergic Chemicals Corporation (Farmingdale, N.Y.) or Spectrum Chemical Manufacturing Company (Gardena, Calif.). The 1995 prices of purified zeaxanthin from these specialty manufacturers, in synthetic racemic mixtures that contain large amounts of the undesirable S-S and S-R isomers, ranges from about $90 to about $125 per milligram (which translates to about $100,000 per gram). Clearly, zeaxanthin is not a widely available chemical, and is not available to the public except in extremely small trace quantities, in mixtures of other carotenoids.
Prior art items which describe zeaxanthin production using microbial fermentation include the following:
(1) Courington and Goodwin 1955, which is the earliest known reference describing the production of zeaxanthin by microbes. The bacteria they described reportedly belonged in the genus Flavobacterium; however, as noted below, microbial classification definitions have changed a great deal over the past few decades, and those bacteria probably would not be classified as Flavobacterium under the current nomenclature.
(2) U.S. Pat. No. 3,891,504 (Schocher and Wiss, 1975, assigned to Hoffman LaRoche). This patent described the production of zeaxanthin by microbes from the genus Flavobacter, which were deposited with the American Type Culture Collection and given ATCC numbers 21081 and 21588. As with the Courington and Goodwin 1955 microbes, these microbes would not be classified as Flavobacter today. These cells and their zeaxanthin pigment were fed to chickens, and caused suitable coloration.
(3) U.S. Pat. No. 3,841,967 (Dasek et al, 1974, assigned to Nestle). This patent described the production of zeaxanthin by microbes from the genus Flavobacter; some of their work involved the same strain cited in the Hoffman-LaRoche U.S. Pat. No. ""504 (21081 and 21588), and they also used ATCC strain 11947. This patent, as well as U.S. Pat. No. 3,951,743 (Shepherd et al, 1976, also assigned to Nestle) described certain cell culturing conditions and nutrient media that would be used to increase the quantity of zeaxanthin produced by the cells being cultured.
(4) Two more recent US patents (U.S. Pat. Nos. 5,308,759 and 5,427,783, both invented by Gierhart and assigned to Applied Food Biotechnology, Inc., the same assignee in this current application) describe a strain of bacteria (Flavobacterium multivorum) isolated from a Missouri waterway. These bacteria were discovered to create zeaxanthin without creating substantial quantities of other carotenoids. This is important, because it makes more zeaxanthin available as a pigment, when fed to poultry or fish to give their flesh a darker color. AFB""s wild-type strain of F. multivorum was deposited with the ATCC, and was given ATCC accession number 55238. Because these bacteria generate a certain type of lipid called sphingolipids, the ATCC has provisionally reclassified these bacteria as Sphingobacterium multivorum, which is the name they are listed under in the ATCC""s catalog. However, as of the filing date of this application, the Sphingobacterium name that appears in the ATCC catalog has not yet appeared in either of the reference works which are widely recognized as the official guides to microbial taxonomy: Bergy""s Manual of Systematic Bacteriology, which is appended and updated by the International Journal of Systematic Bacteriology.
Gierhart""s U.S. Pat. No. ""759 claims methods for producing zeaxanthin, using AFB""s F. multivorum bacteria. The U.S. Pat. No. ""783 (which was a divisional) claims feed mixtures that can be given to poultry or fish. Both of these patents are limited to using zeaxanthin in poultry or fish feed; although zeaxanthin is much more expensive than lutein, it is several times more effective (on a per-weight basis) than lutein in imparting color to animal flesh or chicken yolks. Neither of the Gierhart patents says or suggests anything about using zeaxanthin for treating humans.
Various efforts to synthesize zeaxanthin by standard chemical means (without using microbial fermentation) have been reported over the past 20 years (e.g., U.S. Pat. No. 4,153,615, Saucy 1979). However, non-fermentation processes suffer from several disadvantages. They typically require numerous reaction steps, and each step has a less-than-100% yield, so that the final yield of zeaxanthin at the end of the multi-step processing tends to be relatively poor. In addition, chemical synthesis tends to yield undesirable S-S and S-R stereoisomers of zeaxanthin, as well as various conversion products such as oxidized zeaxanthin, and zeaxanthin molecules which have lost one or more of the double bonds in the straight portion or end rings.
Recently, Hoffman-LaRoche obtained two US patents which relate to the chemical synthesis of the R-R isomer of zeaxanthin; these are U.S. Pat. Nos. 4,952,716 (Lukac et al 1990) and 5,227,507 (Lukac et al 1993). These processes require the production and purification of three major intermediates, with yields of approximately 70 to 85% for each intermediate from its precursor. The overall process described in the Lukac et al patents apparently requires a series of 14 reaction steps, which take a minimum of 83 hours (excluding purification), and the synthesis reactions yield a mixture of reactants and products. This reaction mixture must then be extensively treated to purify the R-R isomer of zeaxanthin. Accordingly, the entire process that would be required for both synthesis and purification using this technique would make production on a commercial scale very difficult, and extremely expensive.
Two types of poultry feed additives may be of interest, since they have the highest lutein or zeaxanthin quantities of any commercially available animal feeds. Certain types of poultry feed additives prepared from corn gluten contain a relatively high percentage of zeaxanthin (about 15-30%), when measured as a percentage of total carotenoids. However, the total carotenoid content of these feed additives is very low (only about 100 milligrams of total carotenoids per pound of poultry feed). The other type of poultry feed additive is prepared from marigold extracts. This additive contains roughly 100-200 times as much yellow pigment per pound of additive (i.e., about 10 to 20 grams of lutein and zeaxanthin per pound); however, more than 95% of the yellow pigment in this marigold preparation is lutein, not zeaxanthin. Zeaxanthin comprises only about 2 to 5% of the yellow pigment in this poultry feed additive (Bauernfeind 1981).
As a matter of prior art, it should also be noted many health food stores sell carotenoid preparations that are labelled as being beneficial to the eyes and eyesight. That labelling claim on carotenoid mixtures may be valid and reasonable, since (as noted above) xcex2-carotene and vitamin A are known to be useful and beneficial in the eyes as general anti-oxidants. However, even though at least one commercially available carotenoid mixture that is sold in health food stores (the xe2x80x9cBeta-Carotene Formula Preparation,xe2x80x9d sold by General Nutrition Corporation) lists zeaxanthin as one of the carotenoids contained in their carotenoid mixtures, none of the commercially available carotenoid mixtures contains more than extremely small, trace quantities of zeaxanthin. The great majority of the carotenoids in the carotenoid mixtures that are sold in health food stores are other, non-zeaxanthin carotenoids (mainly xcex2-carotene and vitamin A).
The current positions and publicly stated research goals of several government agencies and research consortia are also worth noting. The National Institutes of Health, acting through the National Eye Institute (NEI) and the National Advisory Eye Council, has issued two recent government publications which are directly relevant. These two reports are xe2x80x9cVision Research: A national Plan 1994-1998,xe2x80x9d NIH Publication No. 93-3186 (1994; see pages 55-65 in particular), written by the members of the National Advisory Eye Council, and xe2x80x9cAge related eye disease study,xe2x80x9d NIH Publication 93-2910 (1993). Both publications, and the research projects they describe, focus on xcex2-carotene rather than zeaxanthin as the compound which holds the greatest apparent promise for treating AMD. To the best of the Applicants"" knowledge and belief after discussing the subject with officials of the NEI, neither the NEI nor any other organization affiliated with the National Institutes of Health is willing to fund, or has recently funded, any research on zeaxanthin as a potential treatment for AMD. Instead, the NIH and various other tax-funded organizations are allocating millions of dollars to carry out research on xcex2-carotene as the most promising candidate agent for treating or preventing AMD.
Another research group that deserves attention (and which contains various members who are employed as researchers at the NEI) is the Eye Disease Case Control Study Group. This group recently published two articles entitled, xe2x80x9cAnti-oxidant status and neovascular age-related macular degeneration,xe2x80x9d Arch. Ophthalmol. 11: 104-109 (1993), and xe2x80x9cRisk factors for neovascular age-related macular degeneration,xe2x80x9d Arch. Ophthalmol. 10: 1701-1708 (1992). As in the official NIH reports, neither of these articles teaches or suggests the use of zeaxanthin as a drug for treating AMD. To the best of the Applicants"" knowledge and belief, this consortium also has declined or refused to fund any research into zeaxanthin as a possible agent for treating or preventing AMD.
Accordingly, as of late 1995, there are no available sources of concentrated or purified zeaxanthin for human use, either as a drug or as a nutritional supplement.
One object of this invention is to disclose an economically viable method of producing zeaxanthin pills (such as capsules or coated tablets) which contain the R-R stereoisomer of zeaxanthin as the sole detectable zeaxanthin isomer in the formulation, and which are intended and well-suited for human ingestion as a prescription drug or as a nutritional supplement, to help treat or prevent macular degeneration.
Another object of this invention is to disclose food preparations such as margarine, dairy products, syrup, cookie dough, meat preparations that will not be subjected to harsh cooking conditions, and other similar foodstuffs, which contain the R-R stereoisomer of zeaxanthin as the sole detectable zeaxanthin isomer in the formulation, and which are intended for human ingestion, and which can be produced at a reasonable and economically viable cost in commercial quantities.
Another object of this invention is to disclose zeaxanthin preparations that are intended and well-suited for human ingestion as a prescription drug or nutritional supplement, which contain the R-R stereoisomer of zeaxanthin as the sole zeaxanthin isomer in the formulation, and which do not contain any other carotenoids that would compete against zeaxanthin for alimentary uptake and which would therefore reduce the bioavailability of zeaxanthin if present in a mixed carotenoid preparation containing zeaxanthin and other competing carotenoids.
Another object of this invention is to disclose that zeaxanthin preparations produced by fermenting AFB""s strain of F. multivorum (ATCC accession number 55238) or its descendants are exceptionally well suited for human ingestion as a prescription drug or nutritional supplement, for treating or preventing macular degeneration.
Another object of this invention is to disclose that a compound called tertiary butyl hydroquinone, abbreviated as TBHQ and also called 2-(1,1-dimethyl)-1,4-benzenediol, is an especially good stabilizing agent in formulations containing zeaxanthin for human ingestion.
Another object of this invention is to disclose micelle formulations containing the R-R isomer of zeaxanthin, for improved bioavailability.
These and other objects will become more apparent in the following summary and description of the invention.
This invention discloses zeaxanthin preparations which contain the 3R-3xe2x80x2R stereoisomer of zeaxanthin as a sole detectable isomer, packaged in a form that is intended and suitable for oral ingestion by humans as a therapeutic drug or nutritional supplement. Zeaxanthin is a yellow carotenoid pigment found in the macula (in the center of the human retina), which helps protect retinal cells against phototoxic damage. The pure R-R stereoisomer can be prepared by fermenting cells, such as Flavobacterium multivorum (ATCC 55238), which do not create any detectable quantity of the undesirable S-S or S-R isomers, and which do not synthesize any other carotenoids. The R-R isomer can be concentrated, in large quantities and at low cost, into a viscous oily fluid containing about 5 to 20% zeaxanthin, by means of a simple solvent extraction process. This oily fluid can be mixed with a carrier such as vegetable oil and enclosed within a digestible capsule, comparable to a conventional capsule containing Vitamin E. Alternately, a zeaxanthin fluid can be added to various types of foods, such as margarine, dairy products, syrup, cookie dough, and certain types of meat preparations which are not subjected to harsh cooking. Additional purification steps can also be used to purify zeaxanthin to a granular or powdered state which contains nearly pure zeaxanthin. Such processing can be used to create formulations such as ingestible tablets, and particulate formulations that can be added to soups, salads, drinks, or other foods. Preferred stabilizers and anti-oxidants are also disclosed herein. When consumed by humans in any of these modes, the pure R-R stereoisomer of zeaxanthin can help treat and prevent macular degeneration, one of the leading causes of blindness and vision loss, especially among the elderly.